Molecular Origins of Phenotypic Changes in the Obese Microvascular Endothelium

项目总结 肥胖是心血管疾病(CVD)的主要危险因素，如动脉粥样硬化、高血压、2型 糖尿病和中风。尽管有很好的证据表明EC功能障碍存在于大多数形式的CVD中，但 肥胖症形成EC功能障碍的机制仍然知之甚少。进一步 血管内皮细胞的异质性使人们对内皮细胞功能障碍的理解复杂化。 最近的研究表明，在大循环中EC亚型之间存在很大程度的差异 (主动脉)可以在短时间内通过肥胖饮食进一步改变。我们的实验室和其他实验室有 研究表明，微血管系统特别容易受到肥胖引起的功能障碍的影响，是一个关键的预测因素 终末器官损伤。在肥胖小鼠的微血管系统中，我们发现NADPH显著增加 氧化物酶1(NOX1)和组蛋白脱乙酰基酶9(HDAC9)，这两个分子已被证明是重要的 内皮功能障碍和表型改变的贡献者。截至撰写本建议书时， 目前还没有研究阐明EC在微循环中的异质性，更不用说 肥胖对这些人群的影响或者NOX1和HDAC9是否在某些特定的 人口数量高于其他人口。为了及时、经济地进行这些实验，我们开发了 一种AAV方法将AgRP，一种食欲肽，特异性地输送到大脑以诱导过度吞噬和 肥胖。这一新方法与内皮特异性谱系追踪小鼠和内皮特异性 HDAC9基因敲除小鼠将有助于测试我们的中心假设，即肥胖改变EC在 微循环有利于表达NOX1的炎性EC表型。目标1将通过肥胖者定义 内皮细胞特异性谱系追踪模型，转录和表型变化潜在的丢失 肥胖小鼠微循环中的内皮功能。利用单细胞RNA测序技术检测FATE基因定位的内皮细胞 从瘦小鼠和肥胖小鼠中，我们将阐明表达NOX1的内皮细胞的亚型并询问共表达 模式。目的2将验证HDAC9在NOX1和NOX1表达增加中起因果作用的假设 微血管内皮细胞功能障碍和体重减轻逆转HDAC9和NOX1的表达 恢复内皮功能。我们将在体外和体内评估HDAC9是否驱动NOX1的表达 以及它的缺失是否改善了肥胖患者的内皮功能障碍。我们还将调查 新型AgRP AAV对微血管内皮细胞功能的减肥作用 表达AgRP，目的是促进减肥。我们预计这项提案的结果将揭示 肥胖症中内皮功能障碍的新机制，并将为深入了解 改善心血管健康的治疗目标。

PROJECT SUMMARY Obesity is a major risk factor for cardiovascular diseases (CVD) such as atherosclerosis, hypertension, type 2 diabetes and stroke. Although it is well documented that EC dysfunction is present in most forms of CVD, the mechanisms by which obesity shapes the development of EC dysfunction remains poorly understood. Further complicating the understanding of EC dysfunction is the heterogeneity of ECs throughout the vasculature. Recent studies have demonstrated a large degree of divergence between EC subtypes in the macrocirculation (aorta) that can be further altered with obesogenic diets over a short period of time. Our lab and others have shown that the microvasculature is particularly susceptible to obesity induced dysfunction and is a key predictor of end-organ damage. In the microvasculature of obese mice, we have shown a striking increase in NADPH oxidase 1 (NOX1) and histone deacetylase 9 (HDAC9), two molecules that have been shown to be important contributors to endothelial dysfunction and phenotypic changes, respectively. As of the writing of this proposal, no studies have been conducted to elucidate the heterogeneity of EC in the microcirculation, not to mention the effect of obesity on these populations or whether or not NOX1 and HDAC9 are preferentially expressed in certain populations over others. To perform these experiments in a time and cost effective manner, we have developed an AAV approach to deliver AgRP, an orexigenic peptide, specifically to the brain to induce hyperphagia and obesity. This novel approach in conjunction with endothelial specific lineage tracing mice and endothelial specific HDAC9 knockout mice will facilitate testing of our central hypothesis that obesity alters EC heterogeneity in the microcirculation to favor inflammatory EC phenotypes expressing NOX1. Aim 1 will define, through an obese endothelial specific lineage tracing model, the transcriptomic and phenotypic changes underlying loss of endothelial function in the microcirculation of obese mice. Using single cell RNA sequencing of fate mapped ECs from lean and obese mice, we will elucidate the subtypes of ECs expressing NOX1 and interrogate co-expression patterns. Aim 2 will test the hypothesis that HDAC9 has a causal role in increased expression of NOX1 and microvascular endothelial dysfunction and that weight loss reverses the expression of HDAC9 and NOX1 and restores endothelial function. We will assess in vitro and in vivo, whether HDAC9 drives the expression of NOX1 and whether its deletion ameliorates endothelial dysfunction in obesity. We will also investigate the effects of weight loss on microvascular endothelial function using a novel floxed AgRP AAV that enables cessation of AgRP expression with the goal of promoting weight loss. We anticipate the findings of this proposal will reveal new mechanisms contributing to endothelial dysfunction in obesity and will provide insight into potential therapeutic targets to improve cardiovascular health.